Methods and compositions for preventing concatemerization during template-switching
Compositions and methods for performing a template-switching reaction are provided that may include reducing or eliminating concatemerization of the template-switching oligonucleotide (TSO). In some embodiments, the composition may comprise: a reverse transcriptase; a TSO that includes a recognition sequence for a site-specific double strand nucleic acid cleaving enzyme, wherein the TSO has at its 3′ end at least one nucleotide capable of hybridizing to at least one or more non-templated nucleotides added to a templated cDNA strand by the reverse transcriptase; and a site-specific double strand nucleic acid cleaving enzyme that cleaves the TSO at the recognition sequence.
1. A composition comprising:
(a) a reverse transcriptase;
(b) a template-switching oligonucleotide comprising a recognition sequence for a site-specific double strand nucleic acid cleaving enzyme, wherein the template-switching oligonucleotide has at its 3′ end at least one nucleotide that can hybridize to one or more non-templated nucleotides added to a templated cDNA strand by the reverse transcriptase; and
(c) a site-specific double strand nucleic acid cleaving enzyme that recognizes the recognition sequence in the template-switching oligonucleotide.
2. The composition of claim 1 , further comprising a RNA template.
3. The composition of claim 2 , wherein the RNA template is total RNA or polyA + RNA.
4. The composition of claim 2 , wherein the RNA template is RNA from a mammal.
5. The composition of claim 2 , wherein the RNA template comprises a 5′ modified GMP cap.
6. The composition of claim 5 , wherein the 5′ modified GMP cap is a 7-methylguanosine or an affinity tag-labeled GMP cap.
7. The composition of claim 1 , further comprising a cDNA synthesis primer which is an oligo(dT) primer, a random primer, or a transcript-specific primer.
8. The composition of claim 1 , further comprising a cDNA synthesis primer having a 5′ tail that does not hybridize to the RNA template.
9. The composition of claim 1 , wherein the template-switching oligonucleotide is a DNA-RNA chimera.
10. The composition of claim 1 , wherein the template-switching oligonucleotide comprises an amplification primer sequence, and optionally a molecular barcode sequence, between the recognition sequence for the site-specific nucleic acid cleaving enzyme and the at least one or more nucleotides at its 3′ end for hybridizing to non-template nucleotides at the 3′ end of the cDNA strand.
11. The composition of claim 1 , wherein the template-switching oligonucleotide comprises three riboguanine residues at its 3′ end.
12. The composition of claim 1 , wherein the reverse transcriptase is Molatney murine leukemia virus (M-MLV), M-MtV reverse transcriptase lacking RNaseH activity, human T-cell leukemia virus type I (HTLV-I) reverse transcriptase, bovine leukemia virus (BIN) reverse transcriptase, Rous sarcoma virus (RSV) reverse transcriptase, or human immunodeficiency virus (HIV) reverse transcriptase, or a variant thereof.
13. The composition of claim 1 , wherein the site-specific double strand nucleic acid cleaving enzyme is a restriction endonuclease.
14. The composition of claim 1 , wherein the restriction endonuclease is active at a temperature in the range of 40° C. to 55° C.
15. The composition of claim 1 , wherein the site-specific double strand nucleic acid cleaving enzyme is an N-glycosylase.
16. The composition of claim 1 , wherein the site-specific double strand nucleic acid cleaving enzyme is a lyase (N-glycosylase-lyase).
17. The composition of claim 1 , wherein the site-specific double strand nucleic acid cleaving enzyme is an endonuclease.
18. The composition of claim 1 , wherein the site-specific double strand nucleic acid cleaving enzyme is a cas9 ortholog.
19. The composition of claim 1 , wherein the site-specific double strand nucleic acid cleaving enzyme is an Argonaut ortholog.
20. The composition of claim 14 , wherein the restriction endonuclease is capable of inactivation by incubation at a temperature of at least 65° C. for a period of 10 minutes.
21. A method for making cDNA comprising:
(a) obtaining a reaction mix comprising the composition of claim 1 , a cDNA synthesis primer and an RNA template; and
(b) incubating the reaction mix so as to produce cDNA molecules that comprise, at the 3′ end, an added sequence comprising the complement of a sequence at the 5′ end of the template-switching oligonucleotide;
wherein, during the incubation of (b), the site-specific double stranded nucleic acid cleaving enzyme prevents concatamers of the double stranded form of the template-switching oligonucleotide at the 3′ end of the cDNA molecules.
22. The method of claim 21 , wherein step (b) comprises incubating the reaction mix at a temperature of 35° C. to 55° C. for a period of at least 30 minutes.
23. The method of claim 21 , further comprising, after step (b), inactivating the reverse transcriptase and the site-specific nucleic acid cleaving enzyme by incubating the reaction mix at a temperature of at least 65° C. for a period of at least 10 minutes.
24. The method of claim 21 , further comprising: (c) amplifying the cDNA molecules to produce amplification products.
25. The method of claim 24 , wherein (c) is performed using a first primer and a second primer, wherein the first primer hybridizes to the added sequence at the 3′ end of the cDNA and the second primer hybridizes to the complement of a sequence in the cDNA synthesis primer; and wherein the amplification is performed by PCR.
26. The method of claim 21 , wherein the method further comprises sequencing the amplification products.
27. The method of claim 21 , wherein the RNA template is from a clinical sample.
28. The method according to claim 21 , wherein the reaction mix comprises a crowding agent.
29. The method according to claim 28 , wherein the crowding reagent is polyethylene glycol.
30. The composition of claim 1 , comprising a crowding agent.
31. The composition and methods according to claim 30 , wherein the crowding agent is polyethylene glycol.